Download Detection of bluetongue virus and African horseslckness virus in co

Onderstepoort Journal of Veterinary Research, 62:217-222 (1995)
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Detection of bluetongue virus and African horseslckness
virus in co-Infected cell cultures with NS1 gene probes
E. H. VENTER1, H. HUISMAN$2 and A.A. VAN DIJK3*
ABSTRACT
VENTER, E.H., HUISMANS, H. & VAN DIJK, A.A. 1995. Detection of bluetongue virus and African
horsesickness virus in co-infected cell cultures with NS1 gene probes. Onderstepoort Journal of Veterinary Research, 62:217-222
The serogroup specificity of the bluetongue virus (BTV) NS1 and VP3 gene probes was confirmed by
means of northern blot hybridization. Under high-stringency conditions both probes hybridized to 22
BTV serotypes (18 South African serotypes, BTV3 from Cyprus and BTV16 from Pakistan) but not to
serotypes that originate from Australia and India. Furthermore, NS1 gene probes of BTV and African
horsesickness virus (AHSV) were used in a dot-spot in situ hybridization procedure to differentiate between BTV and AHSV in co-infected cell cultures. The method detects viral RNA directly in glutaraldehyde-fixed infected cell cultures without prior nucleic-acid extraction or purification. AHSV could be
detected in cells infected with AHSV at a multiplicity of infection of 10-4 PFU/cell in the presence of a
hundredfold excess of co-infecting BTV. The method may have an application in epidemiological surveys to detect different orbiviruses in the same Culicoides population.
Keywords: African horsesickness virus, AHSV, bluetongue virus, BTV, detection, NS1 gene probes,
VP3 gene probes
INTRODUCTION
Outbreaks of bluetongue and African horsesickness
in susceptible sheep and horse populations, respectively, can have severe economic implications. These
viral diseases are transmitted biologically by biting
midges of the Cu/icoides genus (Du Toit 1944) and
are endemic in South Africa. Rapid and accurate detection of these viruses is important for efficient control and epidemiological surveillance. Several different Orbivirus serogroups and/or serotypes of the same
serogroup can co-circulate in Culicoides populations
* Author to whom correspondence is to be directed
1
Department Veterinary Tropical Diseases, University of Pretoria, Onderstepoort, 0110 South Africa
2
Department of Genetics, University of Pretoria, Pretoria, 0001
South Africa
3
Onderstepoort Veterinary Institute, Onderstepoort, 0110 South
Africa
Accepted for publication 5 September 1995-Editor
(B.J. Erasmus & E.M. Nevil1995, personal communication), complicating cell-culture isolation of all the
viruses present in such mixed infected Culicoides
populations.
Bluetongue and African horsesickness viruses (BTV
and AHSV) represent two of the serogroups of the
Orbivirus genus of the Reoviridae family (Knudson
& Shope 1985). Virions of BTV and AHSV have ten
double-stranded RNA-genome segments encapsidated in a double-layered protein capsid comprised
of seven structural proteins (VP1-7), while three nonstructural proteins (NS1, NS2 and NS3) are synthesized in infected cells (Huismans & Van Dijk 1990).
Hybridization experiments in which cloned genome
segments were used as probes, established that
genome segments that encode proteins NS1 and
VP3 of BTV, AHSV, equine encephalosis virus (EEV)
and epizootic haemorrhagic disease virus (EHDV)
are well conserved within their respective serogroups
(Huismans & Cloete 1987; Bremer, Huismans & Van
217
Detection of bluetongue virus and African horsesickness virus
Dijk 1990; Venter, Van Dijk, Huismans & Vander Lugt
1992; Viljoen & Huismans 1989; Nel & Huismans
1990). In a dot-spot in situ hybridization procedure that
detects virus-specified RNA directly in glutaraldehyde-fixed infected cell cultures without prior nucleicacid extraction or purification (Paeratakul, De Stasio
& Taylor 1988), the NS1 probes detected viral RNA
significantly earlier than those of any of the other conserved genes (Venter, Viljoen, Nel, Huismans & Van
Dijk 1991 ). In the case of BTV and EHDV, it has been
shown that the advantage of the NS1 probe is due
to the fact that the NS1 gene is transcribed at a higher frequency than are the other genome segments
(Huismans & Verwoerd 1973; Huismans, Bremer &
Barber 1979).
In this paper the use of the NS 1 gene as group-specific probe for detecting replicating BTV and AHSV is
further investigated, with particular emphasis on seragroup-specificity and the possibility of detecting viruses in cell cultures co-infected with BTV and AHSV.
MATERIALS AND METHODS
Viruses
All viruses were obtained from Dr B.J. Erasmus
(Onderstepoort Biological Products). The BTV seratypes were virulent viruses propagated in cell cultures, with the exception of serotype 10, which was
an egg-attenuated avirulent strain . The geographical origins of the different BTV serotypes are summarized in Table 1. The viruses were propagated by
limited passaging in monolayer BHK-21 cells (Huismans 1979). The titres of inoculums were determined
on monolayers of Vero cells and expressed as
plaque-forming units per m~ (PFU/m~) (Howell, Verwoerd & Oellermann 1967).
Northern-blot hybridization of dsRNA from
different serotypes
Viral dsRNA was purified from infected BHK-cell cultures as described by Bremer (1976). Two hundred
nanograms dsRNA of each of the 24 BTV serotypes
was fractionated by electrophoresis on 4% polyacrylamide gels (Loening 1967). 32 P end-labelled BTV4
dsRNAserved as a control size marker. After electrophoresis, dsRNA was denatured by soaking the gel
in 50 mM NaOH, transferred electrophoretically to a
Hybond-N membrane (Amersham) and fixed by
exposure to UV light, before hybridization to genomic
probes (Maniatis, Fritsch & Sambrook 1982).
Dot-spot in situ hybridization technique
Confluent monolayers of BHK-21 cells were infected
with BTV or/and AHSV at the multiplicity of infection
(m.o.i.) indicated in the text. Viruses were adsorbed
to the cells for 45 min at 3rC, after which the cells
218
TABLE 1 Origin of the different BTV isolates
BTV serotypes
Country of origin
1, 2,4,5,6, 7, 8, 9, 10, 11, 12, 13
14, 15, 18, 19, 22, 24
South Africa
3
Cyprus
16
Pakistan
17
USA
20, 21
Austral ia
23
India
were rinsed once with serum-free Eagle's medium,
followed by further incubation at 3r C. At different
periods after infection the cells were harvested by
trypsination, collected by low-speed centrifugation
and suspended in Eagle's medium. Hundred-microlitre volumes of appropriate dilutions of cells were filtered onto Hybond-N membranes (Amersham), previously wetted with double-distilled water by means
of a 96-well manifold-filtering apparatus (Bio-Rad).
Cells were fixed on membranes in a solution containing 1% glutaraldehyde, 3% NaCI, 10 mM NaH2 PO
and 40 mM Na2 HPO (pH 7,4) at 4 °C for 1 h, and
rinsed three times with 50 mM EDTA, 100 mM TrisHCI (pH 8,0) proteolytic buffer, containing proteinase
K, at a concentration of 20 ~g/m~ for 30 min at 37 oc,
as described by Paeratakul eta/. (1988). The filters
were briefly air-dried and prehybridized for at least
4 h at 42 °C, in a buffer containing 50% formamide,
1 X Denhardt's solution, 4 X sse buffer, 0,02 M Naphosphate buffer (pH 6,8), and 0,1 mg/m~ of herringsperm DNA.
End-labelling of dsRNA
Unfractionated BTV4 dsRNA was radio-actively
labelled to produce size markers for electrophoresis,
with the use of cytidine 3 ' ,5 '- [5 '-32 P] biphosphate
([ 32 P]pCp) triethylam monium salt and RNA ligase
(England & Uhlenbeck 1978; England, Bruce & Uhlenbeck 1980). 32 P-Iabelled RNA was separated from
unincorporated labelled nucleotides by Sephadex
G-75 column chromatog raphy. The specific activity
of the labelled RNA was 1 x 107 cpm/~g .
Preparation of
32 P
radio-labelled probes
DNA probes were labelled with 32 P-dCTP (1 000 Ci/
m~) by nick-translation of cloned segments of the VP3
and NS1 genes of BTV and the NS1 gene of AHSV.
A commercially available BRL kit was used (Huismans & Cloete 1987; Bremer, Huismans & Van Dijk
1990). The specific activity of probes was generally
in the order of 5 x 107 cpm/IJQ DNA.
E. H. VENTER, H. HUISMANS & A.A. VAN DIJK
RESULTS AND DISCUSSION
Hybridization
Nylon membranes containing fixed dsRNA and cells,
were hybridized to the different genomic probes in a
hybridization mixture containing 50% formamide, 1
X Denhardt's solution, 4 X sse buffer, 0,02 M Naphosphate buffer (pH 6,8), and 0,1 mg/mQ of herringsperm DNA. The probes were boiled and immediately cooled before they were added to the hybridization solution. Hybridization was performed for 16 h
at 42 °C, after which membranes were washed under stringency conditions requiring 90% similarity for
hybridization, namely in a buffer containing 0,1%
SDS and 0,1 x SSC at 65
(four to five times for
15 min each). Autoradiography was carried out by
exposure of the membranes to Cronex MRF X-ray
film (Dupont, USA) at -70°C, with the use of an intensifying screen.
Specificity of the BTV4 NS1 and VP3 gene
probes
Since some reservations have been expressed about
the use of dot-spot hybridization to determine the genetic relatedness of cognate RNA segments of different BTV serotypes (Unger, Chuang, Chuang, Doi &
Osburn 1988), northern blot hybridizations were performed to verify the dot-spot results of Huismans &
Cloete (1987) in respect of the serogroup specificity
of the NS1 and VP3 gene probes. The data depicted
in Fig. 1 confirms that both genes are conserved within the BTV serogroup. Under conditions requi ring
more than 90% nucleic-acid-sequence simi larity,
both probes hybridized to the 18 South African seretypes, BTV3 from Cyprus and BTV16 from Pakistan.
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FIG. 1 A. Autoradiograph of the hybridization of a 32 P-Iabelled BTV4 NS1 -specific DNA probe to a northern
blot ofdsRNAofthe 24 serotypes of BTV. The size marker (C BTV4) is BTV4 end-labelled dsRNA
B. Autoradiograph of the hybridization of a 32 P-Iabelled BTV1 0 VP3-specific DNA probe to a northern blot ofdsRNAofthe 24 serotypes of BTV. The size marker (C BTV4) is BTV4-Iabelled dsRNA
2 19
Detection of bluetongue virus and African horsesickness virus
Neither probe reacted with serotypes 20 and 21 which
originate from Australia. In addition, the VP3 probe
did not react with serotype 23 from India. The sensitivity of the northern blot appears to be lower than
that of the dot-spot hybridization procedure, where
faint signals were detected with serotypes 20 and 21
with both probes. There is a general correlation in the
relative intensity of the hybridization signals between
the NS1 and VP3 gene probes of the different seratypes (Fig. 1A & 1 B) , indicating that the variation in
the intensity of hybridization signals among the seratypes largely reflects differences in the nucleotide
sequence of the cognate genes. The resu lts are also
in agreement with the dot-spot hybridization results
of Huismans & Cloete (1987) and the results of Gould
& Pritchard (1990) , who carried out a limited
A
BTV PROBE: CO-INFECTED CELLS
BTVPFU/cell
10-6
AHSV PFU/cell
1Q-4 1Q-4
1()-4
1Q-3
BTV PROBE: INDIVIDUALLY INFECTED CELLS
10-2
10-1
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AHSV PROBE: CO-INFECTED CELLS
AHSV PROBE: INDIVIDUALLY INFECTED CELLS
BTV PFU/cell
10-6
1()-4
10-3
1Q-2
10-1
AHSV PFU/cell
10-4
1()-4
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FIG. 2 A. Autoradiograph of the hybridization of BTV4 NS1 probe
B. Autoradiograph of the hybridization of AHSV3 NS1 probe to cells infected with BTV4 at a m.o.i. varying from 1 x 10- 5-1
PFU/cell and AHSV3 virus at a m.o.i. of 1 1 PFU/cell as indicated above the lanes. The cells were harvested at 36 h p.i.
o-
220
E.H. VENTER, H. HUISMANS & A.A. VAN DIJK
nucleotide-sequence comparison of conserved
genes of isolates of the same BTV serotypes. Their
results indicated that the variation in nucleotide sequence within a particular geographical region is less
than that of regions which are far apart. Specifically,
nucleotide sequences of conserved cognate genes
of South African and Australian BTVs were reported
to have a variation of about 20%, while those of
South African and North American BTVs differ by
approximately 10%.
Detection of viral RNA in cells co-infected
with BTV and AHSV
Since RNA probes have been reported to be more
sensitive than DNA probes, a positive- and a negative-sense single-stranded RNA (ssRNA) and a DNA
NS1 gene probe in the in situ dot-spot hybridization
procedure were compared. It was considered that
there was no advantage in using ssRNA probes. The
sensitivity of the negative-sense ssRNA was the
same as that of the DNA probe, and with both ssRNA
probes there was considerably more non-specific
background than with the DNA probe, similar to what
has been reported by Squire, Stott, Dangler & Osburn (1987). The positive-sense ssRNA probe yielded only background signals (data not shown) .
Experiments were carried out to investigate under
which conditions the in situ dot-spot hybridization procedure could be used to differentiate between different orbiviruses in the case of mixed infections. In a first
experiment, monolayers of BHK cells were infected
with AHSV3 at a m.o.i. of 10-4 PFU/cell and co-infected with BTV4, with a m.o.i. range of 1o-5-1 PFU/
cell. Controls included cells individually infected, under the same conditions, with either BTV or AHSV. All
infections were done in sixfold to allow harvesting at
six different time intervals between 16 and 60 h p.i.
Different dilutions of the harvested cells were spotted on duplicate membranes and fixed. The duplicate
membranes were probed with the 32 P-Iabelled BTV4
or AHSV3 NS1-gene DNA probe, respectively (Fig. 2).
Hybridization signals with the AHSV probe were observed from 36 h p.i. onwards, in as few as 1 x 104
cells. The results at later times yielded very little
additional information. No cross-hybridization was
observed between BTV and AHSV when the respective NS1 gene probes were used. As expected, the
detection of AHSV in cells co-infected with BTV,
depended on the ratio of infectious BTV to AHSV
particles. When the cell cultures were infected with
AHSV at a m.o.i. of 10-4 PFU/cell, the virus could be
detected 36-h p.i., even when the cells were co-infected with BTV in a hundredfold excess (m.o.i. of
1o-2 PFU/cell) (Fig. 28). At a BTV m.o.i. of 1o-1 PFU/
cell (a thousandfold excess) or more, BTV completely outcompetedAHSV and no evidence of AHSV replication was observed. No significant increase in the
strength of the AHSV hybridization was observed
when cells were harvested at longer periods p.i. (data
not shown). To investigate the influence of a higher
AHSV m.o.i. on the replication of both AHSV and
BTV in co-infected cells, the above-mentioned experiment was repeated, the investigators using an
AHSV m.o.i. of either 1o-1 or 1o-2 , co-infected with
BTV at the same m.o.i. range as in the previous experiment. The results indicated that at a higher m.o.i.
of AHSV, AHSV could be detected at least 6 h earlier,
p.i. and in the presence of increased amounts of BTV,
provided-as in the previous experiment-that BTV
was not present in more than a hundredfold excess
(data not shown).
The dot-spot in situ hybridization procedure compares well with a rapid and sensitive hybridization
assay in solution, for the quantitative determination
of rotaviral RNA with the use of an RNA probe which
detected rotavirus RNA in 5000 infected cells at 48
h p.i. (Johnson & McCrae 1988). This report is the
first description of conditions under which the dotspot hybridization procedure could be of use in orbiviral epidemiological surveillance for detection and
isolation of different orbiviruses in mixed infected Culiciodes populations.
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